Biochemical and Biological Properties of Myosins

肌球蛋白的生化和生物学特性

• 批准号: 6815657
• 负责人: EDWARD D KORN
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6815657
• 关键词: Acanthamoeba; Aspergillus; Dictyostelium; actins; adenosinetriphosphatase; enzyme activity; myosin light chain kinase; myosins; phosphorylation; point mutation; protein folding; protein isoforms; protein structure function

We are interested in the molecular bases of the regulation of the actin-activated ATPase activities of class-I and class-II myosins, the biological roles of these myosins and the regulation of their biological activities. In one project, to understand the coupled contributions of myosin heads and tails to enzymatic activity and function, we studied the properties of chimeras consisting of the motor domain of Dictyostelium myosin II and the tail domains of either Acanthamoeba or smooth muscle myosin II. The chimeras have 10-15 higher actin-activated ATPase activity than wild-type Dictyostelium myosin II but, in contrast to wild-type myosins, this activity is not regulated by phosphorylation of either the regulatory light chain or, in the Acanthamoeba chimera, phosphorylation of the tail. When expressed in myosin II-null cells, both chimeras rescue two myosin II-dependent functions, cytokinesis in suspension culture and capping of con A receptors, but do not support a third myosin II-dependent function, full development to fruiting bodies. It had previously been shown by others that the tail domain of Dictyostelium myosin II contains all the information needed for localization to the cleavage furrow (CF) of dividing cells. We have now found that the 283-residue assembly domain, the shortest tail segment that assembles in vitro, goes to the CF when expressed in myosin II-null cells, as does also an equivalent 256-residue region from the tail of Acanthamoeba myosin II. Thus, there seems to be no specific sequence requirement for localization of myosin to the CF. We have compared the abilities of 15 mutants of the HCM/TEDS-site surface loop of Dictyostelium myosin II to support the three myosin-II dependent functions. The chicken smooth muscle myosin loop supports normal con A-receptor capping, 80% of normal growth and only slightly impaired development. The human beta-cardiac myosin loop supports growth at only 25% of wild-type rate, incomplete capping and greatly impaired development, i.e. to the the tipped mound stage, only slightly better than myosin II-null cells. Various point mutations make the smooth muscle loop fully functional and the cardiac loop function more like the smooth muscle loop. The actin-activated ATPase and in vitro motility activities of all of the mutants are being studied to see which, if either, correlates with their biological properties. Studies of chimeras in which the heavy chain head, neck and tail domains and light chains of Acanthamoeba and Aspergillus myosin I are interchanged in various combinations provide further evidence for the interdependence of the three heavy chain domains and light chains for myosin activities.

我们感兴趣的是调节I类和II类肌球蛋白的肌动蛋白激活的ATP酶活性的分子基础，这些肌球蛋白的生物学作用及其生物学活性的调节。在一个项目中，为了了解肌球蛋白头部和尾部对酶活性和功能的耦合贡献，我们研究了由网囊藻肌球蛋白II的马达结构域和阿米巴或平滑肌肌球蛋白II的尾部结构域组成的嵌合体的性质。嵌合体具有比野生型网囊藻肌球蛋白II高10-15倍的肌动蛋白激活的ATP酶活性，但是与野生型肌球蛋白相反，这种活性不受调节轻链的磷酸化或在阿米巴嵌合体中尾部磷酸化的调节。当在肌球蛋白II-空细胞中表达时，这两种嵌合体拯救两种肌球蛋白II依赖性功能，悬浮培养中的胞质分裂和Con A受体的封盖，但不支持第三种肌球蛋白II依赖性功能，完全发育为子实体。先前已经有人证明，网囊藻肌球蛋白II的尾部结构域包含了定位于分裂细胞的卵裂沟（CF）所需的所有信息。我们现在已经发现，283个残基的组装域，最短的尾段，在体外组装，去CF表达时，在肌球蛋白II-空细胞，也是一个等效的256个残基区域从尾部的阿米巴肌球蛋白II。因此，肌球蛋白定位于CF似乎没有特定的序列要求。我们比较了15个HCM/TEDS位点表面环的Dictyosteoblastoma肌球蛋白II的突变体的能力，以支持三个肌球蛋白-II依赖的功能。鸡平滑肌肌球蛋白环支持正常的con A受体帽，80%的正常生长和仅轻微受损的发育。人β-心肌肌球蛋白环仅支持野生型速率的25%的生长，不完全的帽化和严重受损的发育，即到尖丘阶段，仅略优于肌球蛋白II-无效细胞。各种点突变使平滑肌环功能完全，心脏环功能更像平滑肌环。肌动蛋白激活的ATP酶和所有突变体的体外运动活性正在研究中，看看，如果有的话，与它们的生物学特性。嵌合体的研究，其中阿米巴和曲霉肌球蛋白I的重链头，颈和尾结构域和轻链在各种组合互换提供了进一步的证据，三个重链结构域和轻链的肌球蛋白活性的相互依赖性。